Rabbit antibodies against the procoagulant activity (VIII:C) of human factor VIII. Some theoretical and practical considerations on the human factor VIII molecule using heterologous antibodies.
The procoagulant activity VIII:C was separated from factor VIII antigen (VIIIR:Ag) by gel filtration in the presence of 0.25 mol/l calcium chloride. Antibodies (anti-VIII:C) were obtained by immunization of rabbits with VIII:C. The last step of the purification procedure of antibodies consists of an adsorption on VIIIR:Ag-Sepharose 2 BCL as immunoadsorbent to remove contaminating traces of antibodies against VIIIR:Ag. The anti-VIII:C titer remains unchanged during this adsorption (29 Bethesda units per mg). In solution, anti-VIII:C neutralizes factor VIII activity (in plasma, cryoprecipitate or in purified form) and the fragment VIII:C without reacting with VIIIR:Ag. Once immobilized on a solid matrix, i.e. 2% agarose, it loses over 95% of its inhibitory capacity. The immobilized anti-VIIIR:Ag binds stoichiometrically the antigen and the activity of plasma factor VIII. These results together suggest that factor VIII is composed of 2 different entities, but undissociated under physiological conditions. Immunophysical analyses as a function of pH and temperature of anti-VIII:C and its complex with factor VIII show properties similar to those of homologous antibodies. The antigen determinants of VIII:C (VIII:CAg) are destroyed at low pHs or high temperatures, and VIII:C can no more form a complex with anti-VIII:C. Purified anti-VIII:C is also used in a two-stage assay to detect VIII:CAg or cross-reacting material in some severe haemophiliacs.
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D005169:Factor VIII / Q000276:immunology*; D006467:Hemophilia A / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008968:Molecular Conformation; D009500:Neutralization Tests; D011817:Rabbits / Q000276:immunology; D013696:Temperature
D000906:Antibodies; D005169:Factor VIII